The Bacillus subtilis sporulation A bacterial model of differentiation Or How to survive

Spore formation in Bacillus subtilis and other gram-positive bacteria B. subtilis is a gram-positive soil-dwelling bacterium that has the ability to differentiate into endospores when severely starved for nutrients. (carbon, nitrogen, and/or phosphorus source). Spores are metabolically dormant and highly resistant to a variety of environmental insults, including UV and gamma radiation, reactive oxygen, high and low temperature, acid and alkali conditions, hydrolytic enzymes, and organic solvents. Spores can remain dormant for long periods of time (perhaps up to 50 years), but will germinate when they encounter a nutrient-rich environment containing one or more of two specific ‘germinants’ (L-alanine and/or a mixture of L-asparagine, fructose, glucose, and potassium ions). Spore formation in B. subtilis is a simple developmental event that involves the generation of two different cell types (forespore and mother cell) with different programs of gene expression and different developmental fates. Thus, in addition to being of interest to microbiologists, spore formation in Bacillus is also of considerable interest to developmental biologists. In addition to B. subtilis, several other species of gram-positive bacteria make endospores. Bacillus anthracis (anthrax), Clostridium botulinum (botulism), Clostridium tetani (tetanus), Clostridium perfringens (gas gangrene, food poisoning), Clostridium difficile (antibiotic-associated and pseudomembranous colitis).

Bacillus subtilis growth and sporulation Relatives: B. cereus, B. anthracis time Log(N) inoculate 4 Sporulate, asymmetric cell div 4 3 Gain genetic competence 3 2 Gain motility 2 1 log growth, symmetric cell division 1 Quorum sensing ~7 hours VII stages Genetics: >200 genes/proteins

Life cycle of Bacillus subtilis B. subtilis can sporulate when the environmental conditions become unfavorable ? division cycle sporulation-germination cycle metabolic and environmental signals Start with a schematic overview of the life cycle of B. subtilis. Use this slide to draw attention to the question mark. This is the important developmental decision.

Microscopy of sporulation

Endospores of Bacillus have been the primary model system DNA complexed with SASPs Dipicolinic acid Brock Biology of Microorganisms, vol. 9, Chapter 3

Stage of sporulation I II III IV to VI (vegetative growth) (vegetative growth) I (axial filament formation) II (polar division) III (engulfment) IV to VI (cortex and coat formation) Mother cell lysis and release of free spore Mutants defective in sporulation that are blocked at each of the above morphological stages have been identified. Nomenclature of mutants: spo; stage of block; locus; allele Example: spo0K141

Sporulation Stages in Bacillus I Replication 0 Growth II Assymetric Septum Sporulation Stages in Bacillus VII Free Spore III Engulfment VI Mother Cell Lysis IV Cortex V Coat

Stages of Sporulation in Bacillus subtilis Germination Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII Normal vegetative growth Engulfment Cortex formation Maturation Asymmetric septation Prespore protoplast Coat formation Release

Spore germination In response to nutrients, the spore will exit its dormant state and enter back into vegetative growth free cell spore

B. subtilis sporulation stages 2 chromosomes, axial fibers I 2 cells, cell wall M D II Engulfment III Double membrane IV Cortex V-VI Coat VII Spore maturation Mother cell lysis (DNA)

Stages of Sporulation in Bacillus subtilis How is the process regulated? Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII spo0 spoII spoIII spoIV spoV spoVI

Stages of Sporulation in Bacillus subtilis Golden age of Bacillus genetics Identify spo genes Clone spo genes Determine their interdependencies Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII spo0 spoII spoIII spoIV spoV spoVI

Deciding to sporulate s s sporulate 3 3 Chromosomes: ? 2 Cell density: 1 1 Starvation: ? s H sporulate What is monitored? [GTP] = (C, N, P) Not ppGpp “quorum sensing” peptides Food low, [cell] low Ok ? Food low, [cell] high Trouble ahead ! : undamaged, complete, and polar placement.

How is the septum placed asymmetrically? Polar Division How is the septum placed asymmetrically?

FtsZ: Ring at septum

A Downward Spiral

Asymmetric Cell Division in B Asymmetric Cell Division in B. subtilis Involves a Spiral-like Intermediate of the Cytokinetic Protein FtsZ Cell 109:257-266.

(from Daniel and Errington, 2003. Cell. 113: 767)

Spo0J and Soj spoIIA Spo0J Soj spoIIE spoIIG Spo0J (a ParB homolog) is required for efficient chromosome segregation during vegetative growth in B. subtilis. Spo0J binds to a series of DNA sites (parS sites) in the origin-proximal 20% of the chromosome, and these sites are believed to be involved in partitioning. In addition to its role in partitioning, Spo0J also is needed for efficient spore formation. Spo0J regulates the initiation of sporulation by antagonizing the function of Soj. SoJ (a ParA homolog), a DNA-binding protein present in an operon with Spo0J, inhibits the initiation of sporulation in the absence of Spo0J. Soj inhibits the onset of sporulation by binding and inhibiting transcription of promoters of sporulation genes that are normally activated by Spo0A~P (spoIIA, spoIIE, spoIIG). Spo0J Soj spoIIE spoIIA spoIIG

Roles of Spo0A-P and the alternative s factor sH in polar cell division Spo0A~P, Spo0H (from Margolin, 2002. Curr. Biol. 12: R391-392). Early in sporulation, FtsZ localization switches from a midcell position to a bipolar localization. Spo0A and the phosphorelay proteins Spo0F and Spo0B are needed for the midcell to bipolar switch of FtsZ rings. A constitutively active form of Spo0A is sufficient to cause polar septa formation during exponential growth. These results indicate that Spo0A~P drives transcription of one or more genes that promote formation of polar septa. The alternative s factor sH (encoded by spo0H) is also needed for the switch in FtsZ localization and for polar septation.

Formation of polar septa during sporulation appears to proceed through an intermediate spiral-like FtsZ structure Spo0A~P: expression of SpoIIE Spo0H: increased FtsZ expression (from Margolin, 2002. Curr. Biol. 12: 391-392).

FtsZ spiral formation and polar septation is mediated by increased transcription of SpoIIE and FtsZ during sporulation Early during sporulation, Spo0A~P drives expression of spoIIE, a gene encoding a phosphatase needed for later events in development. SpoIIE co-localizes with FtsZ, first in a spiral-like structure, and then in polar rings.SpoIIE has also been shown to bind directly to FtsZ. Early in sporulation, transcription of FtsZ increases. Increased transcriptionis mediated primarily by a sH-dependent promoter (P2). Bacterial single mutants in which either spoIIE or the P2 promoter are inactivated make asymmetric septa during sporulation. However,double mutants in which both the spoIIE gene and the P2 promoter of ftsZare deleted are unable to make FtsZ spirals or polar septa .In addition, simultaneous overexpression of both spoIIE and ftsZ result in formation of FtsZ spirals followed by polar septa in growing cells in the absence of sporulation stimuli (e.g. nutrient deprivation). These results indicate that ‘polar switching’ of FtsZ localization is mediated by increased transcription of spoIIE and ftsZ early in development. wt (spoIIE+; P2+-ftsZ) spoIIE+; P2∆-ftsZ spoIIE∆; P2+-ftsZ Three hours after the induction of sporulation, cells were fixed, stained with the membrane dye FM4-64, and examined by fluorescence microscopy (from Ben-Yehuda and Losick, 2002. Cell. 109: 257).

Role of sE in polar septum formation sE (the product of the spoIIGB gene) is synthesized as an inactive precursor before formation of the asymmetric septum. Active (processed) sE is generated only in the mother cell where it directs gene expression in that cell type. Null mutations in spoIIGB or mutations that prevent activation of sE result in formation of ‘disporic’ cells. These are cells that contain two asymmetric septa, one at each pole. Both of the forespore-like compartments in disporic cells contain a chromosome, but the larger mother cell is anucleate. Of course, viable spores do not develop from a disporic cell. The disporic phenotype of spoIIGB mutants indicates that sE must be needed for transcription of a gene or genes whose product(s) somehow act to inhibit formation of a second polar septum. A recent report indicates that at least three of the sE -controlled genes that inhibit the second polar septum are spoIID, spoIIM, and spoIIP. spoIID, spoIIM, spoIIP sE - minus sE - positive

Chromosome segregation during sporulation in B. subtilis (from Pogliano et al. 2003. Curr.Opin. Cell Biol. 6: 586)

Role of RacA in chromosome segregation during sporulation In exponentially growing cells, chromosomes are segregated prior to cell division. In sporulating cells, The chromosome becomes organized into an elongated structure (axial filament). Chromosome segregation occurs after most of the asymmetric septum has formed. Initially, only about 35% of one of the chromosomes is located within the forespore. After septation, The remainder of the chromosome is transferred from the mother cell into the forespore. Recent results indicate that the DNA-binding protein RacA is needed for axial filament formation and for efficient translocation of DNA into the forespore. RacA is localized to the cell poles in sporulating cells, suggesting that it might act to anchor the chromosome to this region. Consistent with this idea, expression of RacA is not expressed in vegetatively growing cells and artificial expression of RacA during exponential growth results in movement of nucleoids towards the cell poles (not shown). racA+ strain racA- strain DAPI (DNA) DAPI + FM4-64 (membrane) DAPI (DNA) DAPI + FM4-64 (membrane) sporulating cells (from Ben-Yehuda et al. 2003. Science. 299: 532)

RacA may cooperate with Spo0J and Soj to anchor chromosomes during sporulation (from Errington et al. 2003. Mol. Microbiol. 49: 1463)

Role of SpoIIIE in chromosome translocation Mutations in spoIIIE cause a defect in chromosome translocation into the forespore, indicating that SpoIIIE is needed for chromosome transport into the forespore. SpoIIIE localizes to the asymmetric (polar) division septum. The carboxyl-terminal region of SpoIIIE is critical for the ability of this protein to promote import of the forespore chromosome. Point mutations in the carboxyl-terminal region cause a defect in chromosome translocation. This region in SpoIIIE is homologous a region in DNA transfer (Tra) proteins involved in conjugation-mediated plasmid transfer in Streptomyces. (from Levin and Losick. p. 167-189. In Prokaryotic Development. 2000. ASM Press. Washington, DC).

Promoter recognition by sigma factors    ' RNA polymerase core enzyme Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

Promoter recognition by sigma factors Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

Promoter recognition by sigma factors Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

Sigma factors in sporulation Old gene names New gene names Protein names Location rpoD sigA 37,A vegetative spo0H sigH H vegetative spoIIG sigE 37,E mother cell spoIIA sigF F forespore spoIIIC sigK K mother cell spoIIIG sigG G forespore

Sigma factors in sporulation H H A A Starvation (and other signals) Stage 0

Sigma factors in sporulation E H F A E F A H A Starvation (and other signals) Stage II/III

Sigma factors in sporulation E H H F A A Starvation (and other signals) Stage 0

Sigma factors in sporulation E E H F F F A E A H A Starvation (and other signals) Stage II/III

Sigma factors in sporulation K G E F A K E G F A Starvation (and other signals) Stage IV Starvation (and other signals) Stage III

Sigma factors in sporulation Sporulation: regulated sigma factor cascade … but what regulates the sigma factors? Not transcriptional control What kind of post-transcriptional control? How are events coordinated? Checkpoints?

Genes required for the initiation of sporulation (spo0 genes) spo0A: Encodes a response regulator and key transcription factor. Spo0A~P binds and activates transcription of promoters of genes required for later events in sporulation. Spo0A-P also represses transcription of the transcriptional repressor AbrB, resulting in increased expression of genes involved in alternative stationary-phase responses (degradative enzyme biosynthesis, motility, antibiotic production, competence development). kinA, kinB, kinC: Code for histidine protein kinases that serve as sources of phosphate for Spo0A. spo0F: Encodes a response regulator that transfers phosphate from KinA, KinB, and KinC to the histidine-phosphoprotein Spo0B. spo0B: Encodes a phospho-transfer protein that serves as a direct phosphate donor to Spo0A.

Biochemical analysis of the phospho-relay (from Burbuyls et al. 1991. Cell. 64: 545)